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10 protocols using aps coated glass slides

1

Mouse IFN-γ cDNA Cloning and In Situ Hybridization

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The cDNA fragments of mouse IFN-γ were amplified by RT-PCR using previously described in situ hybridization primers (Kimura et al., 2006 (link)). The amplified fragments were cloned into the pCR-blunt II-TOPO vector (Invitrogen). Serial sagittal sections, 16 µM thick were mounted on APS-coated glass slides (Matsunami Glass) and processed for in situ hybridization as previously described (Fujiwara et al., 2016 (link)).
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2

Immunofluorescence Staining of Tissue Sections

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artTLOs and lymphoid tissues from recipient mice were embedded in Tissue-Tek OCT compound (SAKURA FINETEK), and snap frozen in liquid nitrogen. Five-micrometer-thick cryostat sections were prepared and placed on APS-coated glass slides (Matsunami Glass Ind. Ltd.). Sections were fixed with cold acetone for 5 min, dried, and kept at −80°C until use. After blocking with 5% normal rat serum and 1% BSA in TBS-T (Tris-buffered saline with 0.005% Tween20) for 1 h at 20°C, sections were incubated for 1 h at 20°C with appropriate antibodies or streptavidin-fluorochrome reagents diluted in blocking buffer and washed with PBS three times every 5 min.
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3

Immunohistochemistry of Mouse Embryos

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E10.5 whole embryos were washed with PBS, fixed in 4% paraformaldehyde at room temperature for 30 min, and rinsed three times with PBS. Whole embryos were cryoprotected in 30% sucrose in PBS overnight at 4°C and then embedded in OCT compound. Cryostat sections (12 µm) were cut and affixed to APS-coated glass slides (Matsunami Glass). The sections were then blocked with PBS containing 5% FBS, 1% albumin, and 0.25% Triton X-100 for 1 h at room temperature. The sections were incubated with primary antibodies overnight at 4°C. Fluorescent marker (Cy2, Cy3, or Cy5)-conjugated secondary antibodies (Jackson Laboratory) were used to label each primary antibody at room temperature for 2 h. Optical sections were viewed using a Zeiss confocal laser scanning microscope (LSM 700) with 20× and 40× objectives. Detailed information for primary antibodies is summarized in Supplemental Table S6.
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4

Histological Analysis of Haustorium Development

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Field-collected samples were washed with tap water to remove soil. Safranin-O staining of whole haustoria was performed as previously described (Yoshida and Shirasu 2009 (link)). For histological sections, haustoria and infected host roots were separated and fixed with FAA solution (10% formaldehyde, 5% acetic acid, 50% ethanol) under vacuum for 15 min followed by incubation at room temperature for 2 h. Samples were subjected to an ethanol dehydration series and embedded into Technovit 7100 resin (Heraeus Kulzer GmbH) according to the manufacturer’s instruction. Solidified resin blocks were mounted on wood blocks with Technovit 3040 (Heraeus Kulzer GmbH), then sectioned using a microtome (Leica, RM2135) to 4 μm thickness. Sections were stained with 0.1% Safranin-O (Sigma-Aldrich) and counterstained with 0.1% Fast Green (Wako Chemical) on APS-coated glass slides (Matsunami Glass).
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5

In Situ Hybridization of Mouse NDPK-D

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The cDNA fragments of mouse NDPK-D were amplified by RT-PCR using the in situ hybridization primers previously described[43 (link)]. The amplified fragments were TA-cloned into the pGEM-T Easy vector (Promega, WI, USA). Serial sagittal sections, 16 μM thick were mounted on APS-coated glass slides (Matsunami Glass, Osaka, Japan) and processed for in situ hybridization as described previously[44 (link)].
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6

Cryosectioning of Frozen Liver Samples for MSI

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Liver samples were collected from PXB mice at necropsy and stored frozen (-70 to -90°C). Frozen liver tissues were cut with a cryostat (CM3050 S, Leica, Nussloch, Germany) into 10 µm thick sections at -20°C. Sections were thaw-mounted onto amino propyl silane (APS) coated glass slides (Matsunami Glass Ind., Ltd., Osaka, Japan) and dehydrated in 50 mL tubes with silica gel for approximately 30 min at room temperature. Two serial liver sections per animal were cut for MSI analysis of S-3100-CA and PPIX. Sections were stored frozen (-70 to -90°C) until MSI analysis.
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7

Investigating APOBEC3G-HBZ Interaction in ATL

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PBMCs from ATL patients were washed and centrifuged onto APS-coated glass slides (Matsunami Glass) by Cytospin (Shandon). Cells were then fixed with 4% paraformaldehyde, permeabilized, and blocked with Duolink Blocking Solution. After that, cells were stained with mouse anti-HBZ antibody (1A10, generated by immunizing C57BL/6 mice with keyhole limpet hemocyanin [KLH]- conjugated HBZ peptide 97-133 [CKQIAEYLKRKEEEKARRRRRAEKKAADVARRKQEEQE]) or mouse anti-Smad3 (7F3, Sigma-Aldrich) and rabbit anti-human APOBEC3G antibody (D9C6Z, Cell Signaling Technology), which were diluted in Duolink Antibody Diluent, followed by incubation with Duolink In Situ PLA Probe Anti-Mouse PLUS and Duolink In Situ PLA Probe Anti-Rabbit MINUS. Next, cells were incubated with Duolink 5× Ligation Buffer and Duolink Polymerase (10 u/µL), followed by incubation with Duolink Amplification Red and Duolink Ligase (1 U µL). The stained cells were mounted with Duolink®In Situ Mounting Medium with DAPI. The cells were imaged using a C2 confocal microscope (Nikon) and analyzed with ImageJ.
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8

Immunofluorescence Staining of Frozen Tissue Sections

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Frozen sections were cut at a 10-μm thickness and mounted on either poly-L-lysine-coated or APS-coated glass slides (Matsunami, Osaka, Japan). The sections were then fixed with ethanol at −20°C for 30 min and washed with phosphate-buffered saline (PBS). The sections were next incubated with 5% normal donkey serum-PBS to block non-specific antibody binding, followed by sequential incubations in primary and secondary antibodies diluted in 5% normal donkey serum-PBS. For nuclear staining, 4'6-damidino-2-phenylindole (DAPI) was added to the secondary antibody solution. The final stained specimens were mounted with Vectashield (Cat. #H-1000; Vector Laboratories, Newark, CA) or handmade mounting medium containing 1,4-Diazabicyclo[2.2.2]octane, and examined under a BX62 microscope equipped with Nomarski differential interference-contrast and epifluorescence optics (Olympus, Tokyo, Japan). Images were captured using a CoolSNAP K4 CCD camera (Photometrics, Tucson, AZ) and MetaMorph software version 6.1 (Molecular Devices, Silicon Valley, CA).
In histochemical controls, the sections were processed in the same way as those incubated with each antibody, except omitting primary antibody from primary antibody solutions; and we confirmed that the labeling detected by each antibody was not observed in these negative control specimens (data not shown).
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9

In Situ Hybridization for NIPSNAP1 in Spinal Cord and DRG

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Sense or antisense NIPSNAP1 riboprobe RNAs were synthesized in vitro using digoxigenin-11-UTP (Roche) with a 700-bp coding region of NIPSNAP1 as template. A sliding microtome was used to prepare spinal cord sections (40 µm) from paraformaldehyde-perfused mice. DRG sections (10 µm) prepared using a cryostat were mounted on APS-coated glass slides (Matsunami). The free-floating spinal cord and DRG tissue sections on glass slides were hybridized overnight with digoxigenin-labeled riboprobes at 65℃ in a solution containing 50% formamide, 4% dextran sulfate, 250 µg/ml salmon sperm DNA, 250 µg/ml yeast tRNA, 1× Denhardt’s solution, 0.2% SDS, 25 mM EDTA, 750 mM NaCl, and 25 mM PIPES. Hybridized sections were then washed with 0.1× SCC after treatment with 20 µg/ml RNase A for 30 min at 37℃. The sections were then reacted with an alkaline phosphate-conjugated anti-digoxigenin antibody, and hybrids were detected using 2-hydroxy-3-naphtoic acid-2′-phenylanilide phosphate (Roche), 4-chloro-2-metylbenzenediazonium hemi-zinc chloride salt (Fast Red TR, Roche), and levamisole (Sigma-Aldrich). Thereafter, the free-floating spinal cord was mounted on glass slides. Digital images were captured using an LSM510 Zeiss laser-scanning confocal microscope equipped with an argon He–Ne laser and the appropriate filter.
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10

In situ Detection of ALSV RNA in Strawberry Tissues

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The in situ hybridization analysis for detection of the RNA of ALSV in strawberry tissues was performed following a previously described protocol44 (link). In summary, immature strawberry fruits (including sepals and receptacles) were fixed in FAA solution (formaldehyde alcohol acetic acid; formalin:ethanol:acetic acid:water = 10:50:5:35, v v−1), dehydrated by serial concentrations of ethanol and lemozol, and embedded in Paraplast Plus (Sigma-Aldrich, St. Louis, MO, USA). Sections prepared at 12 μm thickness were extended on APS-coated glass slides (Matsunami Glass, Osaka, Japan), deparaffinized and dehydrated. Slides were treated with proteinase K, re-fixed and a digoxigenin (DIG)-labelled probe that was complementary to the Vp24 region (nt 2619–3617) of the RNA2 of ALSV was hybridized; alternatively, a DIG-labelled probe complementary to the P1 region (nt 132–1045) of soybean mosaic virus was hybridized as a negative control. Slides were rinsed, then probes were labelled with sheep anti-DIG Conjugated Alkaline Phosphatase (Roche, Basel, Switzerland), rinsed again and stained with chromogenic substrate NBT/BCIP (Roche) to generate dark blue indigo and formazan dyes. The reaction was stopped by dipping the slide in water. Slides were dehydrated, dried and mounted in Entellan New (Merck, Darmstadt, Germany).
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